Optical tweezers are a means of trapping micro particles underwater without contact and non-invasively using laser beams that are converged by an objective of a large numerical aperture. The principle was first announced by Ashkin in 1986 and was then followed by research on practical applications. At present, a variety of related devices and systems are available in the market.
Optical tweezers are a valuable tool in biological and physical research. In actual applications, however, it is often difficult to control and use the optical tweezers to trap micro particles.
Conventional optical tweezers are briefly described below. FIG. 5 shows the optics of optical tweezers.
In FIG. 5, 101 denotes the laser fiber light source, 102 the collector lens (collimating lens), 103 the surface reflecting mirror M1, 104 the convergent convex lens, 105 the focal plane of the imaging lens, 106 the surface reflecting mirror M2, 107 the imaging lens, 108 the objective and 109 the specimen.
Near-infrared rays from the laser fiber light source (101) pass through the collector lens (102) to become a flux of parallel rays, which are reflected by the surface reflecting mirror (103), and then converge on the focal plane of imaging lens (5) (first imaging plane). The illuminating light rays reflected by the surface reflecting mirror (106) turn into a flux of parallel rays as they pass through the imaging lens (107) and then converge on the specimen (109) (surface) via an objective (108).
Transparent particles (e.g., cells, latex beads, silica particles) with a refractive index greater than that of water are trapped by the known principle of laser beam convergence. Conventional optics allow the position of rays convergent on the specimen to be varied by tilting the surface reflecting mirror (103), thus allowing the researcher to trap particles at any desired position.
To simplify the optics, the flux of parallel laser beams is enlarged by a beam expander to fully cover the pupil diameter of the objective. This enables direct illumination of the objective and converges the rays on the specimen's surface. A detailed description of this procedure is omitted here.
Problems are experienced using the conventional optics shown in FIG. 5; beads of under several micrometers in size may not be trapped by the objective, and even when trapping is successful the trapping force sharply decreases when the particles are at a depth of 5 μm or more in the water, eventually resulting in trapping failures.
We investigated the reason why the trapping force deteriorates sharply for particles at depths of 5 μm or more in the water. The major cause of the problem is the increased spherical aberration that increases with the depth of the beads in the water due to refraction occurring at the interface between the water and glass (also called cover glass). The objective itself may also have residual spherical aberrations. To solve these problems, an objective with a specific spherical aberration that cancels other spherical aberrations in the system must be selected or some other suitable means must be taken.
The light pressure against a micro particle in water in the direction opposite to the light source increases with the decreasing angle with the optical axis (according to the law of conservation of momentum), and flicks the particle away (pushes the particle upward under an inverted microscope). This force works contrary to trapping, or in other words, weakens the trapping force.
Another possibly effective means of reinforcing the trapping force is increasing the laser output. When studying biological specimens under a microscope, however, the specimen is often damaged if near-infrared rays on the specimen exceed a few mW. This procedure is therefore not very practical.
To solve these problems, the inventors of the present invention studied the means of reinforcing the trapping force and found that optical systems with excellent trapping force had a spherical aberration in the positive (+) direction, as evidenced in the captured Point Spread Function (PSF) images (Japan Science and Technology Corporation, Apr. 5, 2001; Megumu Shio and Kazuhiko Kinosita CREST Team 13, “Evaluation Report on Water Immersion Objective”).
Based on this knowledge, additional experimental optics where the spherical aberration occurs in any desired direction in the illumination system were constructed and incorporated in the original optics, with the expectation that the spherical aberration on the convergent plane of the objective would be, when properly adjusted, always in the plus (+) direction. If this approach was successful, any objective could be used without the necessity of careful selection, including one with a spherical aberration in the negative (−) direction. As a result of the experiments, the above design of the additional optics proved effective even when spherical aberration in the water proportional to the depth of the beads in the water increased due to refraction at the interface between the water and glass (also called cover glass). The inventors furthermore cut off the light at the center part of the spread light flux area (a 10 to 20% reduction by area) located after the additional optics, and found that the light pressure vector in the direction of flicking the particle away had decreased. The trapping force in the 3-D dimensions also improved substantially.
The present invention is based on the above research. It provides trapping force reinforcing optical system that are not restricted by the need to select particular objectives or use water immersion objectives which have a limited range of numerical apertures. The present invention allows the use of ordinary objectives of a large numerical aperture favorable for fluorescence observation, thereby solving the problems of optics in conventional optical tweezers.
Compared with conventional optical tweezers, the optical tweezers of the present invention considerably reduce harmful near-infrared rays assuming the same trapping force, or considerably reinforce the trapping force assuming the same output. It is possible to provide attachment-type (add-on type) optical tweezers featuring the characteristics of the present invention that can be realized in currently marketed optical tweezers. It is also possible to control the optical tweezers while performing total internal reflection fluorescence microscopy (using the evanescent field).